Activation of calcium-release activated calcium (CRAC) channels is suggested to require re-organisation and clustering of ER-localized STIM1 proteins prior to localisation with, and co-clustering of, plasma membrane Orai1 proteins, which form the CRAC channel pore. Vascular smooth muscle cells were cultured from discarded segments of human saphenous vein, which were obtained with ethical approval. Fluorescence was used to detect intracellular calcium and subcellular dynamics of eYFP-STIM1 and mCherry-Orai1. In the presence of physiological extracellular calcium, platelet-derived growth factor elicited transient followed by sustained elevations of intracellular calcium. As previously reported (Li et al 2011 Br J Pharmacol 164, 382-393), the sustained elevation of calcium was suppressed by Orai1 siRNA or a CRAC channel blocker, showing that CRAC channels were activated. Platelet-derived growth factor did not evoke clustering of eYFP-STIM1 or mCherry-Orai1 unless there was CRAC channel blockade, after which there was re-organisation and co-clustering of eYFP-STIM1 and mCherry-Orai1 in response to platelet-derived growth factor. In the absence of CRAC channel blockade, clustering was routinely observed in response to thapsigargin, the inhibitor of sarco-endoplasmic reticular calcium ATPase (SERCA). The data suggest that physiological STIM1 clustering is suppressed by calcium influx through Orai1 channels. Supported by the Wellcome Trust.